Renaturation method of recombinant human growth hormone inclusion body protein

Abstract

The invention discloses a renaturation method of a recombinant human growth hormone inclusion body protein. The renaturation method comprises the following steps: collecting thalli of escherichia coli, ultrasonically crushing thalli cells, collecting an inclusion body, rinsing the inclusion body, denaturing and dissolving the inclusion body by guanidine hydrochloride, stirring and renaturating the inclusion body protein at a low temperature, and performing membrane filtration. And ultrasonically crushing the thallus cells, collecting the inclusion body, and sequentially and repeatedly rinsing the inclusion body for 2-3 times. Low-temperature stirring renaturation is adopted, operation is easy, convenient and visual, control is easy, it can be ensured that during dilution renaturation, the secondary structure of denatured protein is in a completely-opened state, and the concentration of protein in all positions in renaturation liquid is uniform, so that protein peptide chains obtained after renaturation are folded correctly, and three-level space structures are consistent as much as possible; therefore, the recombinant human growth hormone achieves high activity and low aggregation.

Description

Technical Field

[0001] The invention relates to a refolding method of recombinant human growth hormone inclusion body protein, belonging to the field of human growth hormone. Background Art

[0002] At present, there are three different technical approaches to express recombinant human growth hormone using E. coli: one is extracellular secretion expression, one is intracellular soluble expression, and one is the formation of intracellular inclusion bodies. Each of the three approaches has its own advantages and disadvantages: the recombinant human growth hormone expressed extracellularly has the disadvantages of low expression level and long fermentation cycle, which cannot meet the needs of the market; the recombinant human growth hormone expressed intracellularly soluble has the disadvantages of low expression level, easy degradation and difficult purification; and the formation of intracellular inclusion bodies, although the expression level is very large, has the disadvantages of difficulty in renaturation, low yield and poor stability.

[0003] For the inclusion body administration of recombinant human growth hormone, the industry mainly adopts controlled rate graded renaturation, which means that after denaturation, the denaturant is diluted by controlling the flow rate. By controlling the flow rate and renaturation time, a gradient of different denaturant concentrations is achieved to promote the renaturation of inclusion body protein and maintain its structural stability.

[0004] Although the production cost of controlled-rate graded renaturation is low, it has the disadvantages of uneven local protein concentration in the renaturation solution during graded renaturation, which leads to inconsistent tertiary spatial structure of the protein after renaturation, reduced activity, and easy degradation and aggregation. Summary of the invention

[0005] The technical problem to be solved by the present invention is: how to ensure uniform local protein concentration in the refolding solution during graded refolding, so as to easily obtain a large amount of high-purity and high-activity protein.

[0006] In order to solve the above technical problems, the technical solution of the present invention is to provide a method for renaturing recombinant human growth hormone inclusion body protein, which is characterized in that it includes the following steps: collecting Escherichia coli cells, ultrasonically disrupting bacterial cells, collecting inclusion bodies, rinsing inclusion bodies, denaturing and dissolving inclusion bodies with guanidine hydrochloride, renaturing inclusion body protein with low temperature stirring, and membrane filtering; ultrasonically disrupting bacterial cells, collecting inclusion bodies, and rinsing inclusion bodies are repeated 2-3 times in sequence.

[0007] Preferably, the step of collecting Escherichia coli cells is: centrifuging the Escherichia coli solution in a high-speed centrifuge at 6000-12000 rpm for 10-50 min, discarding the clear liquid, and collecting the precipitated cell solids.

[0008] Preferably, the step of ultrasonically disrupting bacterial cells is: adding 8 to 12 times the volume of purified water to the bacterial cells, placing them in an ultrasonic disruptor, and disrupting the cells in an ice bath at ≦10°C.

[0009] Preferably, the step of collecting the inclusion bodies is: centrifuging at 9000-15000 rpm in a high-speed centrifuge for 10-50 min, discarding the clear liquid, and collecting the inclusion body solid precipitate.

[0010] Preferably, the rinsing of the inclusion bodies is performed by rinsing the inclusion bodies 3-5 times with injection water containing 1% to 3% detergent.

[0011] Preferably, the step of rinsing the inclusion bodies is: transferring the inclusion bodies to a container, adding deionized water containing a detergent and stirring, stirring at ≦20° C. for 2 to 8 minutes;

[0012] Preferably, the detergent is Triton X-100.

[0013] Preferably, the step of denaturing and dissolving the inclusion bodies with guanidine hydrochloride is as follows: adding a 6M guanidine hydrochloride solution containing 20mM Beta-mercaptoethanol at a volume-to-weight ratio of 30 to 80 times the inclusion bodies after repeatedly rinsing the inclusion bodies, stirring for 8 to 16 hours to denature and dissolve the protein; centrifuging at 9000 to 15000 rpm in a continuous high-speed centrifuge for 10 to 50 minutes, collecting the clarified liquid, and discarding a small amount of black precipitate impurities produced.

[0014] Preferably, the step of low-temperature stirring renaturation of inclusion body protein is: preparing a renaturation liquid with a volume of 80 to 120 times that of the clarified liquid of the inclusion body denatured and dissolved by guanidine hydrochloride in a renaturation tank, and controlling the jacket cooling water circulation to maintain the renaturation liquid temperature at 4-10 degrees Celsius; keeping the renaturation tank motor stirring at 10 to 20 rpm / min, pumping the clarified liquid of the inclusion body into the renaturation tank at a pumping flow rate in the range of 1 to 3 L / min; and renaturing at low temperature for 24 to 36 hours.

[0015] Preferably, after the renaturation is completed, the motor of the renaturation tank is kept stirring at 10-20 rpm / min, and a 3.6 M ammonium sulfate solution of 0.2-0.3 times the total volume of the renaturation liquid is added to make the final concentration of ammonium sulfate reach 0.9 M, the flow rate is 2-5 L / min, and stirring is performed for 10-50 minutes; filtering is performed with a hollow fiber membrane with a membrane pore size of 0.2-0.45 microns; and the clarified liquid at the membrane filtration end is collected for later use.

[0016] The present invention adopts low-temperature stirring renaturation, which is not only simple and intuitive to operate and easy to control, but also can ensure that during dilution renaturation, the secondary structure of the denatured protein is in a fully open state, and the protein concentration at each location in the renaturation solution is uniform, so that the renaturated protein peptide chain is correctly folded and the tertiary spatial structure is as consistent as possible, so that the recombinant human growth hormone achieves high activity and low aggregation. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1The present invention is an operation flow chart of a method for renaturing recombinant human growth hormone inclusion body protein. DETAILED DESCRIPTION

[0018] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0019] The present invention provides a method for renaturing recombinant human growth hormone inclusion body protein, such as Figure 1 As shown, it includes the following steps:

[0020] (1) Collecting Escherichia coli cells

[0021] The E. coli solution was centrifuged in a high-speed centrifuge at 6000-12000 rpm (9000 rpm in this embodiment) for 10-50 min (0.5 hour in this embodiment), the clear liquid was discarded, and 100 g of solid precipitate was collected, i.e., the fermented recombinant human growth hormone E. coli cells;

[0022] (2) Broken bacteria

[0023] Add 8 to 12 times (10 times in this embodiment) volume of purified water to the bacterial cells, place them in an ultrasonic disruptor, and disrupt the bacteria in an ice bath at ≤10°C;

[0024] (3) Collecting inclusion bodies

[0025] Centrifuge at 9000-15000 rpm (12000 rpm in this embodiment) in a high-speed centrifuge for 10-50 min (0.5 h in this embodiment), discard the clear liquid, collect 30 g of solid precipitate, and obtain inclusion bodies;

[0026] (4) The precipitate (i.e., inclusion bodies) is transferred to a container, 1% to 3% (2% in this embodiment) of Triton X-100 in deionized water is added and stirred at ≤20°C for 2 to 8 minutes (5 minutes in this embodiment);

[0027] (5) Repeat steps (2), (3) and (4) 2-3 times;

[0028] (6) Inclusion body lysis and denaturation

[0029] After step (5), 20 g of the precipitate (inclusion body) in step (4) is mixed with 6 M guanidine hydrochloride solution containing 20 mM beta-mercaptoethanol at a volume-to-weight ratio of 30 to 80 times (50 times in this embodiment, i.e. 1000 ml) of the inclusion body, and stirred for 8 to 16 hours to denature and dissolve the protein;

[0030] (7) centrifuging at 9000 to 15000 rpm (12000 rpm in this embodiment) in a continuous high-speed centrifuge for 10 to 50 min (0.5 hour in this embodiment), collecting the clarified liquid, and discarding a small amount of black precipitated impurities;

[0031] (8) preparing a refolding liquid of 80 to 120 times the volume (100 times, i.e., 100 L) of the clarified liquid of the inclusion bodies denatured and dissolved by guanidine hydrochloride in a refolding tank, and controlling the jacket cooling water circulation to maintain the refolding liquid temperature at 4 to 10 degrees Celsius (in this embodiment, the refolding liquid temperature is maintained at 8 degrees Celsius);

[0032] (9) The motor of the regeneration tank is kept stirring at 10 to 20 rpm / min (10 rpm / min in this embodiment), and the clarified liquid collected in step (7) through step (8) is pumped into the regeneration tank at a pumping rate of 1 to 3 L / min (1 L / min in this embodiment).

[0033] (10) low temperature renaturation for 24 to 36 hours (in this embodiment, low temperature renaturation for 24 hours);

[0034] (11) After the refolding is completed, the motor of the refolding tank is kept stirring at 10-20 rpm / min (10 rpm / min in this embodiment), and 0.2-0.3 times (0.25 times, i.e. 25 liters) of the total volume of the refolding liquid is added with a 3.6 M ammonium sulfate solution, and the pump flow rate is 2-5 L / min (in this embodiment, the pump flow rate is 3 L / min);

[0035] (12) stirring at 10 rpm / min for 10 to 50 min (0.5 hour in this embodiment);

[0036] (13) Filtration using a Spectrum hollow fiber membrane with a pore size of 0.2 to 0.45 μm (0.2 μm in this embodiment);

[0037] (14) The filtrate from the membrane was collected to obtain 95 liters of clear liquid.

Claims

1. A method for renaturing recombinant human growth hormone inclusion body protein, characterized in that: The method comprises the following steps: collecting Escherichia coli cells, ultrasonically breaking bacterial cells, collecting inclusion bodies, rinsing inclusion bodies, denaturing and dissolving inclusion bodies with guanidine hydrochloride, refolding inclusion body proteins with low temperature stirring, and membrane filtering; ultrasonically breaking bacterial cells, collecting inclusion bodies, and rinsing inclusion bodies are repeated 2-3 times in sequence.

2. The method for renaturing recombinant human growth hormone inclusion body protein according to claim 1, characterized in that: The step of collecting Escherichia coli cells is as follows: centrifuging the Escherichia coli solution in a high-speed centrifuge at 6000-12000 rpm for 10-50 minutes, discarding the clear liquid, and collecting the precipitated cell solids.

3. The method for renaturing recombinant human growth hormone inclusion body protein according to claim 1, characterized in that: The step of ultrasonically disrupting bacterial cells is as follows: adding 8 to 12 times the volume of purified water to the bacterial cells, placing the cells in an ultrasonic disruptor, and disrupting the cells in an ice bath at ≤10°C.

4. The method for renaturing recombinant human growth hormone inclusion body protein according to claim 1, characterized in that: The step of collecting the inclusion bodies is: centrifuging at 9000-15000 rpm in a high-speed centrifuge for 10-50 minutes, discarding the clear liquid, and collecting the inclusion body solid precipitate.

5. The method for renaturing recombinant human growth hormone inclusion body protein according to claim 1, characterized in that: The rinsing of the inclusion bodies is performed by rinsing the inclusion bodies 3-5 times with injection water containing 1% to 3% of detergent.

6. The method for renaturing recombinant human growth hormone inclusion body protein according to claim 5, characterized in that: The steps of rinsing the inclusion bodies are as follows: transferring the inclusion bodies to a container, adding deionized water containing a detergent and stirring, and stirring at ≦20° C. for 2 to 8 minutes; 7. A method for renaturing recombinant human growth hormone inclusion body protein according to claim 5 or 6, characterized in that: The detergent is Triton X-100.

8. The method for renaturing recombinant human growth hormone inclusion body protein according to claim 1, characterized in that: The steps of denaturing and dissolving the inclusion bodies with guanidine hydrochloride are as follows: adding a 6M guanidine hydrochloride solution containing 20mM Beta-mercaptoethanol at a volume-to-weight ratio of 30 to 80 times to the inclusion bodies after repeatedly rinsing the inclusion bodies, stirring for 8 to 16 hours to denature and dissolve the protein; centrifuging at 9000 to 15000 rpm in a continuous high-speed centrifuge for 10 to 50 minutes, collecting the clarified liquid, and discarding a small amount of black precipitate impurities produced.

9. The method for renaturing recombinant human growth hormone inclusion body protein according to claim 1, characterized in that: The steps of low-temperature stirring renaturation of inclusion body protein are as follows: preparing a renaturation liquid of 80 to 120 times the volume of the clarified liquid of the inclusion body denatured and dissolved by guanidine hydrochloride in a renaturation tank, and controlling the jacket cooling water circulation to maintain the renaturation liquid temperature at 4 to 10 degrees Celsius; keeping the renaturation tank motor stirring at 10 to 20 rpm / min, pumping the clarified liquid of the inclusion body into the renaturation tank, and the pumping flow rate is in the range of 1 to 3 L / min; and renaturing at low temperature for 24 to 36 hours.

10. A method for renaturing recombinant human growth hormone inclusion body protein according to claim 9, characterized in that: After the renaturation is completed, the motor of the renaturation tank is kept stirring at 10-20 rpm / min, and a 3.6 M ammonium sulfate solution of 0.2-0.3 times the total volume of the renaturation liquid is added to make the final concentration of ammonium sulfate reach 0.9 M, and the flow rate of the pump is 2-5 L / min, and the stirring is carried out for 10-50 minutes; the filtration is carried out with a hollow fiber membrane with a membrane pore size of 0.2-0.45 microns; and the clarified liquid at the filter end of the membrane filtration is collected for later use.

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